Dopey2 and Pcdh7 orchestrate the development of embryonic neural stem cells/ progenitors in zebrafish

Summary DOPEY2 has been shown to be associated with Down syndrome and PCDH7 might be involved in Rett syndrome and MECP2 duplication syndrome. The mechanism how both proteins play roles in these syndromes are largely unknown. Here, we show that Dopey2 and Pcdh7 balance the proliferation and differentiation of neural stem cells and progenitors during embryonic neurogenesis to generate proper size and architecture of zebrafish brains. Dopey2 and Pcdh7 mutually restricted expression of each other in zebrafish embryos. Dopey2 was responsible for the proliferation of neural stem cells/progenitors, whereas Pcdh7 was responsible for the differentiation of neural stem cells/progenitors. Both proteins were shown to orchestrate the proper development and arrangement of neural cells in zebrafish embryonic brains. The results provide an insight into mechanisms to understand how the embryonic brain is constituted and how developmental defects occur in the brains of patients with Down syndrome, Rett syndrome, or MECP2 duplication syndrome.


INTRODUCTION
DOPEY2 is localized within the chromatin region that is critical for Down Syndrome (DS) and has been considered to be a candidate gene responsible for neuromorphological alterations and mental retardation in DS. 1 Neuromorphological features in DS patients include hypoplasia of the hippocampus, cerebral cortex, larger parahippocampal gyrus, and smaller cerebellum. Infants with DS have been shown to possess abnormal cortex development and delayed brain maturation. They also carry fewer neurons and have a lower neuronal density in their brains. 2,3 An in situ quantitative assessment of gene expression has shown that DOPEY2 is overexpressed more than 50% in the cerebral cortex, cerebellum, and the hippocampus, respectively. This finding is in agreement to the hypothesis that DOPEY2 plays a potential role in functional brain alterations and in the pathogenesis of mental retardation in Down syndrome. 4 However, how DOPEY2 modulates the neural development and brain maturation remains unknown.
Rett syndrome (RTT) is an early onset neurodevelopmental disorder with severe cognitive and physical disabilities caused by mutation in the X-linked gene methyl-CpG-binding protein 2 (MECP2), 5 a ubiquitously expressed transcriptional regulator. Duplication of the MECP2 locus causes MECP2 duplication syndrome (MDS), a severe neurodevelopmental disorder. 6,7 Despite remarkable scientific progress because the discovery of both disorders, the mechanisms of how MECP2 dysfunctions lead to both RTT and MDS is largely unknown. Protocadherin (PCDH) 7 belongs to d-protocadherin family and is predominantly expressed in the brain and heart and highly expressed in neurons and astrocytes. 8 The mRNA level of PCDH7 is down regulated by MeCP2 9,10 and up regulated by the reduction of MeCP2 functions. These observations suggest that PCDH7 might be involved in Rett syndrome and MECP2 duplication syndrome.
Both DOPEY2 and PCDH7 are membrane proteins able to sense and mediate extrinsic cues in cells. Extrinsic cues regularize cell fate specification and differentiation decisions during the embryonic development of brains. 11 The most important extrinsic cues are morphogenic signals which pattern the neuroepithelium into discrete progenitor domains and modulate the proliferation and differentiation of neural stem cells and progenitors to constitute brain structures. [12][13][14][15] Other components of the extrinsic environment of neural stem cells and progenitors, including the surrounding cell types, cell-to-cell interactions, the extracellular matrix, the basal lamina, are also referred to be involved in embryonic and adult neurogenesis in brains. 16,17 Despite this inherent complexity of extrinsic cues, accurate incorporation of signaling must take place within the neural stem cells and progenitors for appropriate development of the embryonic brains. Cellular membrane proteins are the first places to sense extrinsic cues and initiate the process of extrinsic signal integration. 18 Although roles for extrinsic signals in embryonic brain development are well established, our understanding of the members and roles of the membrane proteins in neural stem cells and progenitors during the embryonic neurogenesis is limited. In the present work, we identify two membrane proteins that balance the proliferation and differentiation of the neural stem cells and progenitors for the proper size and neural cell arrangement of brain during zebrafish embryonic development.

Dopey 2 is expressed in neural stem cells/progenitors of mouse and zebrafish embryos
We screened more than 70 monoclonal antibodies generated by the injection of mouse embryonic stem cells (ESC) into mice 19 and identified one antibody against Dopey2 protein on the surface of mouse ESC (data not shown). Immunofluorescence staining showed that Dopey2 antibody stained the cells within the ventricular zone (VZ) and the sub-ventricular zone (SVZ) marked by the expression of Sox2, a marker for neural stem cells and progenitor cells in mouse embryonic brains ( Figure S1A). In addition, the antibody nicely stained cells in zebrafish embryos, as seen in Figure S1B. The staining showed that Dopey2 protein was found on the surface of zebrafish embryonic cells from one-cell-stage of embryos ( Figure S1B). Further detection showed that Dopey2 protein is mainly located in the regions of zebrafish embryonic brains, consistent with the results derived from the staining of mouse embryonic brains ( Figures S1A and 1B). The results also indicate that mouse Dopey2 is the conserved homologue of zebrafish Dopey2 protein.
The temporal and spatial expressing patterns of dopey2 gene is both maternally and zygotically expressed and has high expressing levels in zebrafish embryonic brains (Figures S1C-S1E).
To evaluate the function of Dopey2 in neural cells, we used fluorescence-activated cell sorting approach to isolate Dopey2 + neural cells from the brain of E15 mouse embryos and cultured them in semi-solid medium. The Dopey2 + neural cells formed more colonies under neural culture conditions ( Figure 1). Then we performed a neural colony-forming cell assay, which is an established approach to discriminate neural stem cells from more committed neural progenitors. [20][21][22] Under conditions promoting proliferation, more than 20% of seeded Dopey2 + cells formed tripotential clones ( Figures 1B and 1D). Less than 10% of the Dopey2cells formed tripotential clones. We assessed the frequency of stemness by determining the self-renewal and tripotential capacity of the secondary neurospheres. The number of neural stem cells was 5-fold higher in the Dopey2 + cell population ( Figure 1E). The results indicate that Dopey2 expressed neural cells contain neural stem cells in mouse embryonic brains.

Dopey2 is involved in the brain development in zebrafish embryos
To identify the function of Dopey2 during the embryonic development, an antisense morpholino oligonucleotide (DO MO) complementary to the translational initiation site of zebrafish dopey2 mRNA (Figure S2A) and N-terminal and C-terminal deleted mRNA mutants of dopey2 were injected into zebrafish embryos ( Figure S2B). The results showed that DO MO effectively blocked the expression of Dopey2 protein ( Figures S2A, S2D, andS2E) and induced defective phenotypes in the brains of zebrafish embryos S3A, B, C). The defective brain phenotypes of the morphants were restored by co-injection of capped misRNA (mRNA with mismatched bases) of the zebrafish dopey2 with synonymous mutations at the MO binding sites ( Figure S3C). The embryos injected with truncated dopey2 mutants displayed the highly similar brain phenotypes in zebrafish embryos ( Figure S3C). The results also demonstrate that the Dopey2 mutants with N-terminal or C-terminal deletion display the dominant-negative interfering paralogous (DNIP) of Dopey2 protein and can disrupt the function of wild type Dopey2 protein in vivo. Finally, we generated dopey2 gene mutant zebrafish (DO MUT) using CRISPR/Cas9 system (Figures 2A-2C, and S2C). In embryos injected with DO MO or DO MUT embryos, the level of Dopey2 protein is significantly decreased or diminished ( Figure S2E). DO MUT embryos displayed defective brain phenotypes that highly liked ones caused by injections of DO MO and truncated dopey2 RNAs ( Figure S3D). Injection of DO MO into DO MUT embryos did not display any additional defects, confirming the efficiency and credibility of DO MO (Figures 2A-2C). The results demonstrate that Dopey2 is involved in the brain development in zebrafish embryos.  We measured the growth of neural cells by BrdU incorporation in zebrafish embryos. The number of BrdU labeled neural cells was greatly reduced in zebrafish morphant embryos (Figures S4D andS4G). The detection of H3S10p, one of the markers which labels the growing cells, showed that H3S10p positive neural cells were also dominantly reduced in zebrafish morphants and DO MUT embryos ( Figures 2E, S4E, andS4G). The detection of another marker for growing cells, PCNA (data not shown), confirmed the observations that disturbed Dopey2 function caused the growing defects of neural cells seen in zebrafish embryos. Together, the results indicate that Dopey2 is responsible for the proliferation of neural stem cells and progenitors by preventing the differentiation of neural stem cells and progenitors during the embryonic neurogenesis in zebrafish.

Dopey2 restrains the expression of Pcdh7 protein in zebrafish embryos
To address the mechanisms how Dopey2 modulates the proliferation of neural stem cell/progenitors, we screened protein expression patterns, mostly plasma membrane proteins, in the embryonic brains of zebrafish and identified that expression of Pcdh7, which we have previously identified is highly expressing iScience Article during the differentiation programs of neural stem cells and progenitors, 21 greatly increased in the zebrafish embryos with the dysfunction of Dopey2. Therefore, we continued to confirm Pcdh7 expression after disrupting Dopey2 functions in zebrafish embryos using an anti-human PCDH7 antibody (Figures 3A and 3B and data not shown). We found that knock down of Dopey2 expression or dopey2 gene mutation in zebrafish embryos dramatically increased the Pcdh7 protein levels in zebrafish embryos tested by the western blotting and immunofluorescence ( Figures 3A, 3B, and3F). We started to determine the relationship between Dopey2 and Pcdh7. Immunofluorescence staining showed that both proteins were distributed close to the cellular membrane but not localized together in GL251, HEK293T and U251 cells ( Figure S5A). Consistently, immunoprecipitation results showed that Dopey2 was unable to pull down Pcdh7. Vice versa, Pcdh7 could not pull down Dopey2 protein (Figures S5A andS5B and data not shown). The results indicate that Dopey2 and Pcdh7 proteins are not located in the same complex. Next, we addressed how Dopey2 modulated the expression of Pcdh7 protein. We found that pcdh7b mRNA levels was not altered or was slightly decreased by the knocking down Dopey2 expression in zebrafish embryos ( Figure 3C). It means that Dopey2 is not involved in the transcriptional regulation of pcdh7b gene. Ubiquitination has long been recognized as a key determinator of protein fate as this process tags proteins for proteasomal degradation. Hence, we used an immunoprecipitation approach to test ubiquitin labeled Pcdh7 protein in zebrafish embryos. Indeed, Pcdh7 protein was labeled by ubiquitin and was involved in the proteasomal degradation by ubiquitination ( Figure 3D). After dopey2 genetic mutation, ubiquitin labeled Pcdh7 was greatly

Pcdh7 constrains the expression of dopey2 mRNA in zebrafish embryos
To figure out which zebrafish paralogous pcdh7 gene is conserved to human PCDH7 ( Figure S6A), we screened pcdh7a MOs and pcdh7b MOs in zebrafish embryos and identified that zebrafish pcdh7b MO (P7 MO) knocked down the protein expression detected by the anti-human PCDH7 antibody ( Figures S7A andS8B). Consistently, we noticed that there were no observable alterations in brain development or neural marker expression following the knockdown of pcdh7a MO in zebrafish embryos ( Figures S6B andS6C). Thus, human PCDH7 protein is the conserved homologue of zebrafish Pcdh7b. To tested whether the expression of Dopey2 was altered by Pcdh7b, we used antisense morpholino oligonucleotides complementary to the slicing site (P7 MO) and translation start site (P7 MO-AUG) of zebrafish pcdh7b RNA as well as truncated RNAs of pcdh7bN-terminal and C-terminal deleted mutants and the pcdh7b mutant zebrafish (P7 MUT) generated by CRISPR/Cas9 system to determine the relationship between Dopey2 and Pcdh7 in zebrafish embryos ( Figures S6D, S7B, andS8A). When P7 MO was used to target the splice site, it was found that pcdh7b RNA was not properly cleaved ( Figure S7A), resulting in a decrease in Pcdh7 protein level ( Figure S8B) and neural defective phenotypes in zebrafish embryos ( Figures 4A-4D). Expression of truncated Pcdh7 was also found to cause neural defective phenotypes in zebrafish embryos ( Figures 4B-4D). By injecting P7 MO-AUG, which targets the translational start site of pcdh7b mRNA, and a P7 MO-AUG target site-eGFP recombinant plasmid into zebrafish embryos, it was shown that P7 MO-AUG effectively inhibited the expression of eGFP ( Figure S7B) and caused neural defective phenotypes in zebrafish embryos (data not shown). In P7 MUT embryos, mutations of pcdh7b gene diminished the expression of Pcdh7 protein in zebrafish embryos ( Figure S8B). However, it was observed iScience Article that mutations of pcdh7b gene did not exhibit any observable changes in phenotypes in zebrafish embryos ( Figures 4E, S8C, and S8D). In addition, the injection of P7 MO into P7 MUT embryos failed to produce any observably phenotypic changes in zebrafish embryos ( Figures 4E, S8C, and S8D). Previous studies have discovered that mutations containing PTCs can result in transcriptional compensation, and a genetic compensation effect is concealing the effects of the morpholino reactions. [23][24][25] The facts that injections of P7 MO into P7 MUT embryos do not cause any defective phenotypes indicate that mutations of pcdh7b gene conceal the P7 MO effects in P7 MUT zebrafish embryos, confirming that zebrafish embryonic phenotypes are caused by P7 MO specifically targeting pcdh7b RNA rather than other RNAs. All tests of the approaches demonstrate that pcdh7 morpholino oligonucleotides show nicely the efficiency and credibility to manipulate zebrafish embryos.
We injected pcdh7 morpholino oligonucleotides and mRNA of truncated Pcdh7 mutants into zebrafish embryos to detect dopey2 mRNA expression. The results showed that levels of Dopey2 were dramatically increased in the zebrafish embryos by knocking down Pcdh7 or after expression of the truncated pcdh7 RNA ( Figures 5A-5C), indicating that Pcdh7 participate in the pathway that regulates the generations of dopey2 mRNA. We used chromatin immunoprecipitation (ChIP) assay to test whether Pcdh7 participates in the pathway that regulates the transcription of dopey2 gene. RNA polymerase II (Pol II) transcribes all protein-coding genes. The C-terminal domain (CTD) of the largest subunit of RNA polymerase II consists of multiple heptad repeats (consensus Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7). In general, after the iScience Article formation of the pre-initiation complex (PIC) at the promoter of a gene, RNA polymerase II with an unphosphorylated CTD, which is detected by the antibody 8WG16, initiates the transcription of a gene. 26 Then, the CTD is phosphorylated on Ser5, which is detected by an anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody, for the elongation of gene transcription. 27,28 We performed ChIP to measure the RNAP II states along the dopey2 gene. The results displayed that Pcdh7 knockdown dominantly increased the number of RNAP II with an unphosphorylated CTD and with Ser5 phosphorylated CTD localized within the promoter region and transcription initiating region of dopey2 gene ( Figure 5D). This phenomenon was also observed when morpholino targeting the translation start site was injected into embryos ( Figures S7C  and S7D). We observed that there were no significant changes in dopey2 gene expression or transcription in P7 MUT embryos (Figures 5E-5G). In addition, injections of P7 MO into P7 MUT embryos did not alter dopey2 expression or transcription activation by RNAP II loading, confirming that P7 MO affects dopey2 transcription by targeting the expression of pcdh7b gene (Figures 5E-5G). Together, the results indicate that Pcdh7 is involved in the pathway to restrict the transcription of dopey2 gene in zebrafish embryos.

Pcdh7 is responsible for the differentiation programs of embryonic stem cells and progenitors
To test Pcdh7b functions during the development programs of zebrafish embryos, we firstly analyzed temporal and spatial expression patterns of pcdh7b gene. The results showed that pcdh7b gene was zygotically expressed in zebrafish embryos (Figures S9A andS9B). The embryonic neural cells dominantly carried highly pcdh7b expressing mRNA levels ( Figure S9C). Because of the genetic compensation effects in zebrafish embryos with pcdh7 gene mutants and the pcdh7 morpholino oligonucleotides specifically targeting to the pcdh7b RNA, we used pcdh7 morpholino oligonucleotides to perform experiments in detail and P7 MUT zebrafish to confirm the specificities of results in zebrafish embryos. In zebrafish P7 MO morphant embryos, the expression of nestin and sox2 were dramatically increased when we measured the zebrafish embryos at the stages of 48 hpf, 50 hpf ( Figures 6A and 6B). The immunostaining of Sox2 confirmed that the neural cells with features of stem cells/progenitors were significantly enhanced after the disruption of Pcdh7b functions in the zebrafish embryos ( Figure 6C). The expression of neuron marker gene huc, oligodendrocyte marker gene oligo2, and astrocyte marker gene gfap were dramatically reduced in the morphant embryos ( Figures 6A, 6B, and S9D, and data not shown). Similarly, embryos injected with P7 MO-AUG at 48 hpf led to an elevation of sox2 expression and decreased huc expression in the embryos ( Figure S7E). Injections of pcdh7 morpholino oligonucleotides into P7 MUT zebrafish embryos did not cause any phenotypes, confirming again the specificities of neural phenotypes caused by pcdh7 morpholino oligonucleotides (Figures S8C andS8D). After we injected P7 MO, the distribution of Huc positive neurons were disturbed and greatly decreased in the transgenic zebrafish embryos with Huc-GFP expression in neurons (data not shown, MS1, 4). The detection of H3S10p showed that the number of H3S10p positive neural cells were dominantly increased in zebrafish P7 MO morphant embryos ( Figures 6D and 6E). Pcdh7b overexpression dramatically decreased the H3S10p positive cells in embryonic brains ( Figures 6D and 6E). Together, the results indicate that Pcdh7b is responsible for the differentiation of neural stem cells and progenitors and prevents the proliferation of neural stem cells and progenitors during embryonic neurogenesis, just the opposite of Dopey2 functions during embryonic neurogenesis.

Dopey2 and Pcdh7 are required to modulate the size and architecture of developmental brains in zebrafish embryos
To determine the functional relationship between Dopey2 and Pcdh7, we coinjected DO MO and P7 MO into one-cell embryos. The results turned out that the expression of sox2 and nestin (data not shown) was comparable to ones in the embryos injected with the Ctl MO ( Figures 7A and 7B). DO MO injections decreased the enhanced expression of sox2, nestin and wnt1 by P7 MO (Figures 7A, 7B, and 7E). On the other hand, P7 MO injection reduced the higher expression of huc, gfap and oligo2 caused by DO MO in zebrafish embryos ( Figures 7A, 7C, and 7D, and data not shown). Furthermore, injection of pcdh7 mRNA was unable to increase the enhanced expression of huc, gfap and oligo2 induced by DO MO in zebrafish embryos ( Figure 7A, and data not shown). In return, the injection of dopey2 mRNA also could not alter the expression of sox2 and nestin induced by P7 MO ( Figure 7A). The simultaneous injection of DO MO and P7 MO into DO MUT embryos was found to ease off the effects of increased sox2 expression and decreased huc expression caused by P7 MO (Figures 7A-7C). On the other hand, the simultaneous injection of DO MO and P7 MO into P7 MUT embryos did not mitigate the phenotypes of decreased sox2 expression and increased huc expression caused by DO MO ( Figure 7F) iScience Article does not alter this outcome. However, in the P7 MUT, the presence of a genetic compensation mechanism results in decreased sox2 and increased huc following DO MO injection, which confirms that the phenotype caused by the combination of DO MO and P7 MO is indeed because of the targeted mechanism. Together, the examinations suggest that Dopey2 and Pcdh7 are involved in a similar way to modulate the proliferation and differentiation of neural stem cell/progenitors during the development of embryonic brains.
We visualized the neurons and structures of embryonic brains in the transgenic zebrafish embryos with Huc-GFP expression in neurons by co-injection of DO MO and P7 MO ( Figure 8A, Videos S1, S2, S3, and S4). By knocking down both the expression of Dopey2 and Pcdh7b in embryonic brains, we were able to restore the arrangement of neurons, which was disturbed by the disruption of Pcdh7 function. However, the brain did not reach a similar size when compared to brains in the zebrafish embryos injected with Ctl MO. After Dopey2 knockdown, neuron formation in the brains of zebrafish embryos was highly increased. The sizes of brains were larger at 72 hpf, the end of the embryonic development in zebrafish, in comparison to the control brains. Knocking down Pcdh7b largely reduced neuron formation and affected neuron arrangements in zebrafish embryonic brains, resulted in the smaller size of brains at 72 hpf of zebrafish embryos. Overexpression of Dopey2 or Pcdh7b caused the smaller size brains at 72 hpf ( Figure 8B, Videos S5, S6, and S7). Consistent with the results, overexpression of Dopey2 decreased neuron formation during the

DISCUSSION
Cellular proliferation versus differentiation of neural stem cells and progenitors has been largely investigated. Initially, the embryonic brain begins with primitive neural precursors, neuroepithelial cells, which gives rise to all neurons and macroglia in the brain. 29,30 Then, neuroepithelial cells begin to differentiate into the population of more fate-restricted stem cells/progenitors that signify common stem cells/progenitors for neurons, astrocytes, and oligodendrocytes. 31,32 The common stem cells/progenitors asymmetrically generate neural cells or produce intermediate progenitor populations that transiently increase the progenitor pool before terminally differentiating into specific types of neuronal and glial cells, each with their precise function. 33 To effectively perform the complicated processes in the developmental brain, intrinsic (e.g., transcription factors) and extrinsic (environmental signals) factors synchronize the determination by neural progenitors to continue to undergo proliferation or to differentiation to produce the populations of neurons and glia cells. [34][35][36] Appropriate numbers of inhibitory and excitatory neurons, oligodendrocytes, and astrocytes are required for normal neural functions in animals. Any imbalance in iScience Article the population of specific neurons, astrocytes, and oligodendrocytes can cause neurophysiological, neuropsychological and intellectual disorders. 37 Studies in the embryonic brain of model animals have identified many factors leading to the understanding of the brain development. It has been revealed that the neurons, astrocytes, and oligodendrocytes that compose the mature brain are generated sequentially from neural stem cell/progenitor pools. The balance of proliferation and differentiation of neural stem and progenitor cells during embryonic development is a causal factor to determine the architecture and the size of brains. [38][39][40] The mechanisms how the balance of proliferation and differentiation of neural stem and progenitor cells is maintained remain unknown. In present study, we demonstrate two molecules, which restrain their expression mutually, to balance proliferation and differentiation of neural stem cells and progenitors and to modulate the cellular number and arrangements for the proper size of embryonic brain. The finding provides a novel way to understand how embryonic brain is constituted.
We demonstrated that Dopey2 controls the neural development and brain maturation by maintaining the proliferation of neural stem cells and progenitors and inhibits the neural differentiation during embryonic neurogenesis. Our results indicate when Dopey2 highly expresses in the brain, neural differentiation is suppressed in certain regions, resulting the defective brain maturation, fewer neurons, and lower neuronal density. The appearance phenocopied the neuromorphological features in DS patients. We have also identified that Pcdh7 is responsible for the differentiation of neural stem cells and progenitor and modulates the neural architecture during embryonic development of zebrafish brains. Increased expression of Pcdh7 reduces the proliferation of neural stem cells and progenitors and enhances the neural differentiations. Disruption or increased expression of Pcdh7b functions causes neural defects during embryonic brains. The phenotypes we saw might explain the developmental defects in the brains of patients with Rett syndrome and MECP2 duplication syndrome. Our results indicate both Dopey2 and Pcdh7 are required to balance the proliferation and differentiation of neural stem cells and progenitors during embryonic neurogenesis to generate proper sizes and architectures of brains. iScience Article

Limitations of the study
We showed that Dopey2 and Pcdh7 mutually restrict their expression through different ways. Previous data show that DOPEY2 is involved in the sorting nexin-3 (SNX3) containing complex. 41,42 SNX3 containing complex retrieves Wntless (Wls) from the early endosome back to the Golgi complex, prevents Wls lysosomal degradation and allows Wls to undergo COPI-mediated retrograde transport back to the ER. DOPEY2-associated complex displays an evolutionary conserved endosome-associated membrane remodeling complex. In vivo suppression of DOPEY2 leads to enhanced lysosomal degradation of Wntless. 41,42 The observations suggest that DOPEY2 prevents the lysosomal degradation of proteins. In our present work, we identify that DOPEY2 does not locate with PCDH7 in cells, excluding the hypothesis that lysosomal degradation of PCDH7 mediated directly by DOPEY2. In contrast, Dopey2 is able to modulate the ubiquitination of Pcdh7 and subjects the Pcdh7 protein to the degradation of ubiquitin proteasome system. In present works, we failed to show how Dopey2 is involved in the pathway of ubiquitin proteasome system. The questions need to be further addressed. We showed that Pcdh7 regulates dopey2 RNA expression through modulating RNA polymerase II loading to the promoter of dopey2 gene. After transcription initiates, Pcdh7 is also able to keep the RNA polymerase II for transcription elongation along the dopey2 gene. Many processes and protein complexes, for instance, complexes for DNA and histone modifications, chromatin remodeling complexes, mediator complexes, pre-initiation complex, or DSIF complex, are involved in RNA polymerase II loading to a gene promoter and for the transcription elongation of RNA polymerase II. 26,28,43 In our present work, we did not figure out how PCDH7, a membrane protein, regulates the nuclear affair for the transcription of a specific gene. Future, more in depth approaches are required to identify the pathways and mechanisms as to how the transcription of a specific gene is regulated by PCDH7.

DATA AND MATERIALS AVAILABILITY
All data are available in the main text or the supplemental information.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following: Data and code availability d Raw and analyzed data reported in this paper will be shared by the lead contact upon request.
d This paper does not report original code.
d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Zebrafish maintenance
We used AB lines and Tg (huc:EGFP) lines to conduct experiments. Adult zebrafish (Danio rerio) were raised and maintained according to standard procedures. Embryos were placed in 28.5 C incubator and staged according to standard procedures. All experiments involving the use of animals were conducted in compliance with the approved guidelines. The animal protocols were approved by the Animal Care and Use Committee of West China Hospital, Sichuan University, China.

Establishmen of the mutant zebrafish line
The mutant zebrafish line was generated by CRISPR-Cas9 system. Optimized guide RNA was designed by CHOPCHOP and Benchling. The target sequences of Dopey2 gene in exon1 is 5 0 -GGCCGATCTCA TCTCCTCAC-3 0 . The target sequences of Pcdh7b gene in exon1 is 5 0 -GGTAATGTAGCCGCGGACCT-3 0 . One-cell stage of wild-type zebrafish embryos were microinjected with 1nL sgRNA and Cas9 protein (GenScript) containing nuclear localization signal (NLS) (final concentrations is 250 ng/mL sgRNA and 150 ng/mL NLS-Cas9). After initial screening by T7EI endonuclease (NEB) and Sanger sequencing, the F0 generation was crossed with wild-type zebrafish to obtain the F1 generation. Until they have grown to 1.5 months, the genotyping of each F1 were performed to obtain F1 mutants carrying frameshift mutations. The homozygous mutant F2 was obtained by mating heterozygous F1 fish with the same mutation type. The primers used to detect dopey2 genotype were 5 0 -GACTGGCAGAATAACCTACA-3' (upper primer) and 5 0 -TTACGGCAATAATTGTTTCG-3 0 (lower primer). The primers used to detect the genotype of pcdh7b were 5 0 -TATGAGGACTACAGGCATTG-3' (upper primer) and 5 0 -AGTGGGCAGGAGATAAAG-3 0 (lower primer).

Production of mouse monoclonal antibodies against mouse embryonic stem cells and identification of surface antigens
The Mouse embryonic stem cells cell (mES) lines D3 (ATCC, http://www.atcc.org/) and J2 were cultured on mitomycin C-treated mouse embryonic fibroblasts (MEFs). The mouse embryonic carcinoma (EC) cell line F9 (ATCC, http://www.atcc.org/) was cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco) supplemented with 12% fetal bovine serum (FBS, Hyclone) at 37 C in 5% CO 2 . The procedures to produce mouse monoclonal antibodies against mouse embryonic stem cells and to identify surface antigens of mouse embryonic stem cells were performed. Briefly, mES D3 cells were injected into two-monthsold BALB/c mice for each immunization. Subsequent boosts were done using mES cell suspension with an interval of two weeks between each immunization. One week after the fourth immunization, serum from the immunized mice was collected and tested for immunocytochemical staining of mES cells. Two weeks after the fourth immunization, the mice were injected intravenously with mES cells to boost the immunization. The mice were sacrificed three days after the last boost, and spleen cells were isolated and used to produce hybridomas. Supernatants from the hybridoma cultures were collected. mES cells were cultured and then stained with hybridoma supernatants and a FITC conjugated goat anti-mouse secondary antibody. Positive clones were subcloned by limiting dilution in 96-well plates. The antigens were identified by hybridoma supernatant and Protein A-conjugated resin, following liquid chromatography-tandem mass spectrometry (LC-MS/MS). The membrane proteins of mouse ES cells were detected by immunofluorescence approaches and flow cytometer using the hybridoma supernatants.

5-bromo-20-deoxyuridine (BrdU) Pulse Labeling
Zebrafish embryos were incubated with 0.1 mg/mL BrdU (Sigma) for 2 hours in an incubator when they developed to 22 hpf (hour post-fertilization). When the embryos developed to 24 hpf, the embryos were collected into 1.5 mL EP tubes, 30 embryos per tube, and fixed with 4% paraformaldehyde in a 4 C refrigerator overnight. Prior to immunostaining, zebrafish embryo sections were incubated with HCl (1 N) on ice for 10 minutes to rupture the DNA structure of the labeled cells, then treated with HCl (2 N) for 10 minutes at room temperature and 20 minutes at 37 C. After the acid washes, these embryos were added borate buffer (0.1 M) and incubated at room temperature for 12 min. Zebrafish embryos were then rinsed in PBS/glycine (1 M). Subsequentstep is the same as zebrafish whole-mount immunofluorescence staining.

Co-immunoprecipitation
To investigate whether Dopey2 interacts with Pcdh7 and the changes in ubiquitination of Pcdh7, HEK293T cells or zebrafish embryos were homogenized in IP buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40) containing protease inhibitor cocktail (Thermo Fisher). After centrifugation at 4 C, the supernatant was collected and combined with beads (Invitrogen) and antibody Pcdh7 (Santa Cruz, sc-517042, 1:50 dilution) overnight. After rinsing with wash buffer, subsequent analysis was performed according to the Western blotting method described above. Band intensities were measured with ImageJ software.

Quantitative RT-PCR
Zebrafish embryos were harvested at the desired period and dechorionated. Total RNA was extracted with TRIZOL and treated with TURBO DNase (Invitrogen), and then used to synthesized cDNA with a reverse transcription kit (TAKARA). SYBR green (ROCHE) kit were used for qQPCR. Each sample was tested in triplicate and BioRad CFX Manager software was used for data analysis. The cycle threshold (Ct) value was obtained. The difference between the Ct values of the target gene and the housekeeping gene was expressed as DCt value. The DDCt value was obtained by subtracting the DCt value of control sample from DCt value of the experimental sample. Relativefold changes in gene expression were calculated as 2 ÀDDCt . The sequences of qRT-PCR primers are listed in TableS1.

Chromosome immunoprecipitation assay
200 Morpholino-injected embryos were collected at 48 hpf, and fixed in 1% formaldehyde, then homogenized and sonicated into 300-500 bp chromatin fragments. Chromatin fragments were immunoprecipitated ll OPEN ACCESS